Potential of faeces for non-invasive assessment of Juan Fernandez fur seals and their environment
The Juan Fernandez fur seal (JFFS) is a marine mammal endemic to the Juan Fernandez Archipelago in the Pacific Ocean. The archipelago is a UNESCO Biosphere Reserve and has been identified as one of the eleven irreplaceable priority sites for marine conservation worldwide. As a result of overhunting between the 17, 18 and 19th centuries, the JFFS was severely reduced. Furthermore, it was presumed extinct by the end of the 19th century. Since its "rediscovery" in the early 60s up to current times, the JFFS population have evidenced a steady recovery. Today, it is an icon for local tourism and a great example of population recovery. Since 1995 the Chilean government has decreed a 30-year hunting ban, enabling an impressive recovery. However, the ban on hunting will only last until 2025, and it is unclear what conservation measures will be put in place after this date. Besides intermittent basic censuses, no further monitoring has been done on this species in the last two decades, to the best of my knowledge. Complicated logistics, inaccessibility, lack of funding, and human resources may partially explain the lack of research. However, in the context of the hunting ban coming to an end in less than five years, there is an urgent need to build as much knowledge as possible in a relatively short period to inform policymakers when deciding on the future protection measures for this species. Studying marine mammals such as the JFFS can bring further benefits. For instance, as high trophic organisms, marine mammals play an essential role in nutrient and energy transfer and regulating other species' abundance. Additionally, their long lifespan and extensive fat storage enable the bioaccumulation and biomagnification of liposoluble toxins. These and other characteristics have made marine mammals an increasing target for understanding and monitoring various changes in oceanic ecosystems, including the behaviour and effects of pollutants. However, adequate knowledge about the target species' behaviour and ecology is required to use these animals as marine bioindicators. To establish a baseline for the study and monitoring of the JFFS, I explored the potential of faecal samples as a non-invasive method to obtain diverse information while lowering sampling cost and logistic complexities. Here, I focused on only three of the multiple topics that can be studied from faecal samples. First, I used 16S rRNA amplicon sequencing to characterise the faecal microbiome (Chapter 3). This first faecal microbiome characterisation evidenced a clear separation of the samples into two clusters. Due to the little information available on the species, it was not possible to provide a clear explanation of the pattern observed here. However, diet and sex (associated with prey selection and, therefore, diet) could be possible explanations. On the other hand, the phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) inferred pathways associated with pathogenesis were enriched in cluster 2, which contained only 22 % of the samples. This first insight contributes to understanding the natural microbial diversity in free-free ranging pinnipeds. Next, I used inductively coupled plasma mass spectrometry to evaluate heavy metal exposure (Chapter 4). The results evidenced high levels of Cd and Hg in the JFFS compared to the Antarctic fur seal (AFS), suggesting high exposure. Diet is the most likely source of contamination. Motivated by these results, I analysed Cd in bone samples, evidencing Cd absorption. These samples, however, did not evidence any of the changes usually associated with Cd intoxication in bone, suggesting some degree of adaptation to high levels of this toxic heavy metal. Furthermore, Si levels, an ultra-trace element related to bone health, could be an interesting target for future studies on Cd tolerance in JFFS. Human studies on this topic may also benefit. Finally, in Chapter 5, I focused on optimising a method for collection, storage, and host DNA extraction and amplification of faecal samples. For this optimisation, I targeted the mtDNA control region, five different microsatellite loci and two loci commonly targeted for molecular sex identification. Swabbing the faecal surface was usually associated with less specific PCR products. However, by performing nested PCRs, the specificity of the amplification dramatically improved in samples with poor DNA. On the other hand, when using more sensitive assays such as real-time PCR, which was used for molecular sex identification, a nested PCR approach should only be considered when direct amplification fails to avoid sample contamination. This study showed that working with faecal samples for investigating population genetics requires significant optimisation. However, once methods become optimised, the difficulty of processing these samples reduce while the probability of success increases. Even though not yet complete, this study is a significant contribution that will enable more rigorous monitoring of the JFFS. The information generated from my research is an essential contribution to the knowledge about this species which is urgently needed to inform policymakers for future conservation policies. Here, I have shown that working with faecal samples can be an accessible alternative to studying various aspects of a species.
Fur seals, Otariid, Microbiome, Host genetics, Heavy metals
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